The growing interest in potential health effects of long-chain polyunsaturated fatty acids (PUFAs) makes it important to evaluate the method used to assess the fatty acid intake in nutrition research studies. We aimed to validate the questionnaire-based dietary intake of selected PUFAs: eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), α-linolenic acid (ALA), linoleic acid (LA), and arachidonic acid (AA) within the Danish National Birth Cohort (DNBC), by comparing 345 women’s reported intake with concentration of plasma biomarkers. The applied questionnaire- and biomarker data reflect dietary intake from around the same time point in mid-pregnancy and relationships were investigated by use of Pearson and Spearman correlation and linear regression statistics. We demonstrated moderate but consistent adjusted correlations between dietary intake estimates and the corresponding plasma biomarker concentrations (differences in plasma concentration per 100 mg/day greater intake of 0.05 (95% CI: 0.02; 0.08)) and 0.05 (95% CI: 0.01; 0.08) percentage of total plasma fatty acids for EPA and DHA, respectively). The associations strengthened when restricting the analyses to women with ALA intake below the median intake. We found a weak correlation between the dietary intake of ALA and its plasma biomarker with a difference in plasma concentration of 0.07 (95% CI: 0.03; 0.10) percent of total plasma fatty acids per 1 g/day greater intake, while the dietary intake of LA and AA did not correlate with their corresponding biomarkers.